The present invention relates to genes for detecting Pectinatus frisingensis or Pectinatus cerevisiiphilus of the genus Pectinatus, which is known as beer-spoilage bacteria, and a method for detecting the bacteria by using the genes.
Bacteria of the genus Pectinatus have been known as beer-spoilage bacteria. In the genus, two kinds of Pectinatus frisingensis and Pectinatus cerevisiiphilus have been known. For detecting the bacteria of the genus Pectinatus, the bacteria must be isolated after multiplication culture and separation culture. It takes at least seven days. Then, isolated bacteria are multiplied and tested by many qualitative tests such as morphological observation, gram stainability, a catalase test, utilization of various carbon sources and the like to identify the bacteria.
These tests are very troublesome, and it takes much time and it costs much. In addition to these common identification tests, there is a method that DNA is extracted from isolated bacteria, fixed on a membrane, and conducted a hybridization test by using standard bacteria DNA as a probe to identify the class. However, it takes some days, and it is difficult to obtain necessary detective sensitivity and selectivity.
Lately, a method for detection of bacteria of the genus Pectinatus is disclosed by using a monoclonal antibody that specifically reacts with Pectinatus cerevisiiphilus (ASBC Journal: 51(4)158-163, 1993). However, the method is insufficient to the detective sensitivity. The method has a problem that Pectinatus frisingensis can not be detected.
The other detection method has been reported. It can detect Pectinatus frisingensis and Pectinatus cerevisiiphilus by a Ribotyping method that polymorphism of a ribosomal RNA gene is detected (J. Am. Soc. Chem.: 56 (1) 19-23, 1998). However, since the method needs operation for isolating the bacteria, it has problems of detective sensitivity and speed.
Considering these problems, further quick detection methods have been studied. W097/20071 discloses a method for detecting Pectinatus comprising extracting DNA of the test microorganism, and using a PCR method that a complementary oligonucleotide of the DNA functionates as a primer. However, the base sequences of 16S rRNA gene used in the technique are sometimes similar to those of microorganisms of the other genera, so that there are problems that the other microorganisms are detected in addition to particular microorganisms to be detected.
The gene in a spacer between a 16S rRNA gene and a 23S rRNA gene has a specific gene sequence. Though methods for detecting microorganisms using the gene sequence are disclosed in Japanese Jozo Ronbunshu 50, 22-31 (1995), APPL. ENVIRON. MICROBIOL. VOL.62, NO.5, 1683-1688(1996), FEMS MICROBIOL LETT. VOL. 84, NO.3, 307-312(1991), Japanese Patent Kokai Publication No. 6-98800 and the like, gene sequences of the spacers of the genus Pectinatus have not been found.
The present invention aims to provide gene sequences of a spacer region that is constituted between a 16S rRNA gene and a 23S rRNA gene specific for the genus Pectinatus relating to beer-spoilage, and to provide a method for sensitively and quickly detecting the genus by using the sequences.
(1) The first invention is a gene sequence of a spacer region between a gene coding 16S rRNA and a gene coding 23S rRNA of Pectinatus frisingensis containing a part of the base sequence or the whole base sequence represented by SEQ ID NO: 1.
(2) The second invention is a gene sequence of a spacer region between a gene coding 16S rRNA and a gene coding 238 rRNA of Pectinatus frisingensis containing a part of the base sequence or the whole base sequence represented by SEQ ID NO: 2.
(3) The third invention is a gene sequence of a spacer region between a gene coding 16S rRNA and a gene coding 23S rRNA of Pectinatus cerevisiiphilus containing a part of the base sequence or the whole base sequence represented by SEQ ID NO: 3.
(4) The fourth invention is a gene sequence of a spacer region between a gene coding 16S rRNA and a gene coding 23S rRNA of Pectinatus cerevisiiphilus containing a part of the base sequence or the whole base sequence represented by SEQ ID NO: 4.
(5) The fifth invention is an oligonucleotide characterized in that the gene sequence of a spacer region between a gene coding 16S rRNA and a gene coding 23S rRNA of Pectinatus frisingensis has at least one of the following sequence group or the corresponding complementary sequence:
5xe2x80x2-CCATCCTCTTGAAAATCTC-3xe2x80x2{circle around (1)} (SEQ ID NO:5)
5xe2x80x2-TCTCRTCTCACAAGTTTGGC-3xe2x80x2{circle around (2)} (SEQ ID NO:6)
(6) The sixth invention is an oligonucleotide characterized in that the gene sequence of a spacer region between a gene coding 16S rRNA and a gene coding 23S rRNA of Pectinatus cerevisiiphilus has at least one of the following sequence group or the corresponding complementary sequence:
5xe2x80x2-CACTCTTACAAGTATCTAC-3xe2x80x2{circle around (3)} (SEQ ID NO:7)
5xe2x80x2-CCACAATATTTCCGACCAGC-3xe2x80x2{circle around (4)} (SEQ ID NO:8)
5xe2x80x2-AGTCTTCTCTACTGCCATGC-3xe2x80x2{circle around (5)} (SEQ ID NO:9)
(7) The seventh invention is a method for detecting Pectinatus frisingensis, wherein the oligonucleotide made from the gene sequence described in (1) or (2) uses as a primer for synthesis of nucleic acids, and the nucleic acid is treated by gene amplification to detect the bacteria.
(8) The eighth invention is a method for detecting Pectinatus cerevisiiphilus, wherein the oligonucleotide made from the gene sequence described in (3) or (4) uses as a primer for synthesis of nucleic acids, and the nucleic acid is treated by gene amplification to detect the bacteria.
(9) The ninth invention is a method for detecting Pectinatus frisingensis, wherein the oligonucleotide made from the gene sequence described in (1) or (2), or the oligonucleotide made from the gene sequence described in (5), and a nucleotide sequence coding 16S rRNA gene of Pectinatus frisingensis use as primers for synthesis of nucleic acids, and the nucleic acid is treated by gene amplification to detect the bacteria.
(10) The tenth invention is a method for detecting Pectinatus cerevisiiphilus, wherein the oligonucleotide made from the gene sequence described in (3) or (4) or the oligonucleotide made from the gene sequence described in (6), and a nucleotide sequence coding 1 6S rRNA gene of Pectinatus cerevisiiphilus use as primers for synthesis of nucleic acids, and the nucleic acid is treated by gene amplification to detect the bacteria.
(11) The eleventh invention is a method as in (9), wherein the nucleotide sequence coding the 16S rRNA gene of Pectinatus frisingensis has the following sequence:
5xe2x80x2-CGTATCCAGAGATGGATATT-3xe2x80x2{circle around (6)} (SEQ ID NO:10)
(12) The twelfth invention is a method as in (10), wherein the nucleotide sequence coding the 16S rRNA gene of Pectinatus cerevisiiphilus has the following sequence:
5xe2x80x2-CGTATGCAGAGATGCATATT-3xe2x80x2{circle around (7)} (SEQ ID NO:11)